Multiple sclerosis (MS) is a debilitating disease and is one of the leading causes of non-traumatic neurological disability among the young population in North America and Europe. The clinical progression of the disease is variable with ˜85% of patients at diagnosis exhibiting unpredictable and recurring episodes of neurological deficits that spontaneously subside. Over time, MS patients endure a slow, progressive and irreversible neurological decline, which may be delayed with early therapeutic intervention. The neurodegeneration and ensuing axonal loss in MS patients results in permanent clinical deficits, including limb paralysis, vision loss, spinal cord symptoms, and cognitive deficit. [Noseworthy J H, Lucchinetti C, Rodriguez M, & Weinshenker B G (2000) Multiple sclerosis. The New England journal of medicine 343(13):938-952] Because of the long duration of disability and high prevalence among young adults, MS is an enormous public health issue with high socio-economic burden and significant impact on the quality of life.
The pathophysiology of MS is thought to have an autoimmune origin with multifocal lesions in the Central Nervous System (CNS) that are characterized by inflammation, demyelination, and axonal injury. This is compounded by the pronounced loss of neuronal connections, which lack the innate capacity to self-repair and an increased susceptibility of adult CNS neurons to apoptotic cell death. It is accepted that irreversible axonal and neuronal loss is a major determinant of the progressive and permanent neurological impairment in MS patients. [Noseworthy J H, Lucchinetti C, Rodriguez M, & Weinshenker B G (2000) Multiple sclerosis. The New England journal of medicine 343(13):938-952] The heterogeneity in the pathology and clinical progression of MS has led to numerous etiological hypotheses, but none have yet to be confirmed. Furthermore, it remains unknown whether neurodegeneration precedes the autoimmune attack in MS or vice versa.
Of interest, elevated circulating iron levels and deposition of iron in the brains of MS have been reported. [Stankiewicz J M & Brass S D (2009) Role of iron in neurotoxicity: a cause for concern in the elderly? Current opinion in clinical nutrition and metabolic care 12(1):22-29; LeVine S M, Bilgen M, & Lynch S G (2013) Iron accumulation in multiple sclerosis: an early pathogenic event. Expert review of neurotherapeutics 13(3):247-2501 Two genes FPN1 (encodes an iron exporter protein) and HEPC (encodes hepcidin, an enzyme that is crucial in iron regulation) were found to increase the incidence of MS by more than 4-fold and 2.5-fold respectively. [Gemmati D, et al. (2012) Polymorphisms in the genes coding for iron binding and transporting proteins are associated with disability, severity, and early progression in multiple sclerosis. BMC medical genetics 13:70] Furthermore, reducing levels of iron resulted in reduced disease severity. [Grant S M, Wiesinger J A, Beard J L, & Cantorna M T (2003) Iron-deficient mice fail to develop autoimmune encephalomyelitis. The Journal of nutrition 133(8):2635-2638; Stankiewicz J M, Neema M, & Ceccarelli A (2014) Iron and multiple sclerosis. Neurobiology of aging 35S2:S51-S58; Weigel K J, Lynch S G, & Levine S M (2014) Iron chelation and multiple sclerosis. ASN neuro 6(1):e00136; LeVine S M & Chakrabarty A (2004) The role of iron in the pathogenesis of experimental allergic encephalomyelitis and multiple sclerosis. Annals of the New York Academy of Sciences 1012:252-266] Notably, MS patients undergoing treatment show decreased levels of iron as compared to patients receiving placebo, thus implying a possible role for iron in mediating disease progression. [Pawate S, Wang L, Song Y, & Sriram S (2012) Analysis of T2 intensity by magnetic resonance imaging of deep gray matter nuclei in multiple sclerosis patients: effect of immunomodulatory therapies. Journal of neuroimaging: official journal of the American Society of Neuroimaging 22(2):137-144] The role of iron in MS remains speculative with no clear understanding on whether iron deposition is the result of neurodegeneration or whether it contributes to the development of the disease.
The identification of the MHC II risk allele in MS patients alluded to a central role played by cluster of differentiation (CD4+) T cells in the development of MS. Analyses of blood and CSF from MS patients further suggested that the disease implicated the recruitment of auto-reactive CD4+ T lymphocytes from the periphery to the CNS, where they tethered, rolled, and adhered to endothelial cells lining the blood vessels. [Friese M A, Schattling B, & Fugger L (2014) Mechanisms of neurodegeneration and axonal dysfunction in multiple sclerosis. Nature reviews. Neurology 10(4):225-238] The subsequent infiltration of these cells to the parenchyma is associated with breakdown of the blood brain barrier (BBB). [Gaitan M I, et al. (2011) Evolution of the blood-brain barrier in newly forming multiple sclerosis lesions. Annals of neurology 70(1):22-29] The initial infiltration of CD4+ effector cells of the T helper 17 (Th17) or Th1 subtypes lead to the secretion of pro-inflammatory cytokines, such as IL-17a and IFN-γ. [Kebir H, et al. (2007) Human TH17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation. Nature medicine 13(10):1173-1175] These cytokines are cytotoxic and stimulate the recruitment of other immune cells, such as CD8+ effector T cells, which are able to further secrete cytotoxic cytokines or antigen-presenting cells, such as CD11c+ cells, which further prime and activate effector T cells within the CNS. This immune cascade together with the activation of CNS resident microglia releases cytotoxic cytokines and reactive oxygen or nitrogen species to damage the network of supporting oligodendrocytes. In addition to this aberrant immune activation, MS patients display a decreased ability to negatively regulate effector T cells, further impacting this immune activation. [Bettelli E, Korn T, & Kuchroo V K (2007) Th17: the third member of the effector T cell trilogy. Current opinion in immunology 19(6):652-657; Viglietta V, Baecher-Allan C, Weiner H L, & Hafler D A (2004) Loss of functional suppression by CD4+CD25+ regulatory T cells in patients with multiple sclerosis. The Journal of experimental medicine 199(7):971-979] Axonal damage occurs early in demyelinating lesions, which correlate highly with infiltration of immune cells. [Ferguson B, Matyszak M K, Esiri M M, & Perry V H (1997) Axonal damage in acute multiple sclerosis lesions. Brain: a journal of neurology 120 (Pt 3):393-399]
The complexity of MS is highlighted by the heterogeneity of pathological patterns that occur in MS patients. These pathological hallmarks are subdivided in four distinct patterns. The initial pathological damage is predominantly regulated by the infiltration of T and B cells at the site of plaque formation (patterns I and II). Typically these pathological patterns coincide with the novel occurrence of plaques. Subsequently, there is a shift towards decreased cellular infiltration and increased neurodegeneration represented by sites of hypoxic insult leading to neuronal death and apoptosis (patterns III and IV). MS was originally thought to target white matter tissue, however extensive gray matter lesions have been identified in the early phases of MS progression. In the progressive stages of the disease, BBB breakdown does occur, but to a lesser extent in comparison to relapsing-remitting (RRMS). Furthermore, immune cell infiltrates are present in areas that maintain BBB permeability. [Lassmann H, van Horssen J, & Mahad D (2012) Progressive multiple sclerosis: pathology and pathogenesis. Nature reviews. Neurology 8(11):647-656] This suggests that in progressive stages, immune activation takes place within the CNS independently of peripheral infiltration. At this stage of the disease, patients suffer from extensive brain atrophy and dilatation of ventricles.
In MS, loss of BBB integrity occurs early in the disease progression. Breakdown of the BBB primes tissue for the recruitment of leukocytes and subsequent neuronal damage. While the BBB normally sequesters immune cells outside of the CNS, it can also promote the penetration of immune cells to localized regions of inflammation within the CNS. Early in MS, endothelial cells (ECs) can enhance leukocyte infiltration by up-regulating both E- and P-selectin proteins on their membranes. [Engelhardt B & Ransohoff R M (2005) The ins and outs of T-lymphocyte trafficking to the CNS: anatomical sites and molecular mechanisms. Trends in immunology 26(9):485-495] Furthermore, ECs can secrete leukocytes attractants such as chemokine ligand 2 (CCL2). [Biernacki K, Prat A, Blain M, & Antel J P (2001) Regulation of Th1 and Th2 lymphocyte migration by human adult brain endothelial cells. Journal of neuropathology and experimental neurology 60(12):1127-1136] Following activation by inflammatory cytokines, ECs can express adhesion molecules, such as intracellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1, which enhance the extravasation of leukocytes through ECs. [Alvarez J I, Katayama T, & Prat A (2013) Glial influence on the blood brain barrier. Glia 61(12):1939-1958]
In the Experimental Autoimmune Encephalomyelitis (EAE) animal model, which is an accepted animal model of MS, invading immune cells most prominently target the spinal cord replicating the pathological patterns I and II of MS. [Rangachari M & Kuchroo V K (2013) Using EAE to better understand principles of immune function and autoimmune pathology. Journal of autoimmunity 45:31-39] The adoptive transfer of CD4+ T cells from immunized mice into naïve mice confirmed that EAE was also a CD4+ T cell-mediated disease. [Paterson P Y (1960) Transfer of allergic encephalomyelitis in rats by means of lymph node cells. The Journal of experimental medicine 111:119-136] Initially, IFN-γ producing Th1 effector cells were believed to mediate the disease, as adoptive transfers of Th1 cells induced EAE in mice. However, the discovery that the induction of EAE was in fact dependent on a novel cytokine, IL-23, led to the identification of a novel CD4+ T cell subtype, the IL-17 producing Th17 cells. [Cua D J, et al. (2003) Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 421(6924):744-748; Becher B, Durell B G, & Noelle R J (2002) Experimental autoimmune encephalitis and inflammation in the absence of interleukin-12. The Journal of clinical investigation 110(4):493-497; Park H, et al. (2005) A distinct lineage of CD4 T cells regulates tissue inflammation by producing interleukin 17. Nature immunology 6(11):1133-1141] Thus, both Th1 and Th17 CD4+ T cell subsets can mediate EAE. Importantly, both IFN-γ and IL-17 secreting cells were shown to be enriched in the CSF of MS patients. [Kebir H, et al. (2007) Human TH17 lymphocytes promote blood-brain barrier disruption and central nervous system inflammation. Nature medicine 13 (10): 1173-1175; Traugott U & Lebon P (1988) Multiple sclerosis: involvement of interferons in lesion pathogenesis. Annals of neurology 24(2):243-251] In EAE, both subtypes of T cells have been shown to promote differential immune cell recruitments, with Th1 promoting monocytic inflammation and Th17 promoting neutrophilic infiltrates. [Kroenke M A, Carlson T J, Andjelkovic A V, & Segal B M (2008) IL-12- and IL-23-modulated T cells induce distinct types of EAE based on histology, CNS chemokine profile, and response to cytokine inhibition. The Journal of experimental medicine 205(7):1535-1541] In addition, atypical forms of EAE may arise from a shift in the expression of these pro-inflammatory cytokines. Higher expression of IL-17 is associated with more prevalent brain lesions whereas it may be protective in the spinal cord. [Stromnes I M, Cerretti L M, Liggitt D, Harris R A, & Goverman J M (2008) Differential regulation of central nervous system autoimmunity by T(H)1 and T(H)17 cells. Nature medicine 14(3):337-342] These studies raise the possibility that T cell subsets may mediate differential functions depending on their tissue localization.
In addition to the well-established role of CD4+ T-cells in the development of EAE, studies have also identified other subsets of T cells that play a crucial role in EAE. Adoptive transfers of CD8+ T cells induce an atypical form of EAE with lesions localized to white matter of the cerebellum. In addition, this model induces wide-scale oligodendrocyte death resembling patterns III and IV of MS patients. [Bitsch A, Schuchardt J, Bunkowski S, Kuhlmann T, & Bruck W (2000) Acute axonal injury in multiple sclerosis. Correlation with demyelination and inflammation. Brain: a journal of neurology 123 (Pt 6):1174-1183] Recent findings highlight the occurrence of multiple subsets of CD8+ T cells present in both EAE and MS. Initial studies using CD8−/− knockout mice have shown increased severity of the disease and more frequent relapses, indicating a possible regulatory role of CD8+ T cells in EAE. [Jiang H, Zhang S I, & Pernis B (1992) Role of CD8+ T cells in murine experimental allergic encephalomyelitis. Science 256(5060):1213-1215; Koh D R, et al. (1992) Less mortality but more relapses in experimental allergic encephalomyelitis in CD8−/− mice. Science 256(5060):1210-1213] Furthermore, CD8+/CD28−/− T cells have been shown to induce immunosuppressive phenotypes in EAE by interrupting co-stimulatory molecule expression on the surface of CD4+ T cells. [Montero E, et al. (2004) Regulation of experimental autoimmune encephalomyelitis by CD4+, CD25+ and CD8+ T cells: analysis using depleting antibodies. Journal of autoimmunity 23(1):1-7]
Although T cells have been shown to mediate disease, B cells could also be involved in the pathophysiology of EAE. MS patients possess higher IgG levels in the CSF compared to age-matched controls, suggesting the presence of antibody-releasing cells within the CSF. Studies have found that B cells enhance EAE severity by promoting differentiation of Th1 and Th17 cells. Prior to EAE onset, a regulatory subset of B cells, able to regulate immune response, exist. This may provide a novel biomarker from which the shift of B cells from regulatory to pathogenic may correlate with MS onset. [Fillatreau S, Sweenie C H, McGeachy M J, Gray D, & Anderton S M (2002) B cells regulate autoimmunity by provision of IL-10. Nature immunology 3(10): 944-950]